Abstract

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase
with a structure composed of a kinase domain related to those of
LIM-kinases and a unique C-terminal proline-rich domain. Like
LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both
in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated
the formation of actin stress fibers and focal adhesions. In contrast
to LIM-kinases, the kinase activity of TESK1 was not enhanced by
Rho-associated kinase (ROCK) or p21-activated kinase, indicating that
TESK1 is not their downstream effector. Both the kinase activity of
TESK1 and the level of cofilin phosphorylation increased by plating
cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited
LIM-kinase-induced cofilin phosphorylation but did not affect
fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa
cells. Expression of a kinase-negative TESK1 suppressed cofilin
phosphorylation and formation of stress fibers and focal adhesions
induced in cells plated on fibronectin. These results suggest that
TESK1 functions downstream of integrins and plays a key role in
integrin-mediated actin reorganization, presumably through
phosphorylating and inactivating cofilin. We propose that TESK1 and
LIM-kinases commonly phosphorylate cofilin but are regulated in
different ways and play distinct roles in actin reorganization in
living cells.

INTRODUCTION

Actin cytoskeletal reorganization plays important roles in many
basic cell activities, including cell movement, adhesion,
morphogenesis, and cytokinesis. Actin reorganization is often triggered
in response to extracellular stimuli, such as binding of growth factors
and chemoattractants to cell surface receptors and ECM proteins to
integrin receptors. To better understand the mechanisms of
stimulus-induced actin reorganization, it is important to elucidate the
signaling pathways that transduce external stimuli to the machinery
controlling the dynamics and organization of actin filaments.

Actin filament dynamics, which underlie the actin reorganization, are
coordinately regulated by several types of actin-binding proteins (Chen
et al., 2000). Among them, cofilin and its close relative,
actin-depolymerizing factor (ADF), bind to actin monomers and filaments
and have the potential to depolymerize and sever actin filaments; hence
they seem to play an essential role in the rapid turnover of actin
filaments (Moon and Drubin, 1995; Bamburg et al., 1999). The
activity of cofilin is reversibly regulated by phosphorylation and
dephosphorylation at Ser-3, with the phosphorylated form being inactive
(Agnew et al., 1995; Moriyama et al., 1996).
Cofilin phosphorylation is stimulated by lysophosphatidic acid in
N1E-115 neuroblastoma cells (Maekawa et al., 1999), whereas
it is down-regulated by thrombin in platelets, chemoattractants in
neutrophils, and other stimuli (Moon and Drubin, 1995). We and
other investigators provided evidence that LIM-kinase 1 (LIMK1) and
LIM-kinase 2 (LIMK2) (Mizuno et al., 1994; Okano et
al., 1995) phosphorylate cofilin specifically at Ser-3, both in
vitro and in vivo, and regulate actin cytoskeletal reorganization by
phosphorylating and inactivating cofilin (Arber et al.,
1998; Yang et al., 1998). LIM-kinases are activated in
cultured cells by Rho family small GTPases, Rac, Rho, and Cdc42, albeit
the activation is indirect (Arber et al., 1998; Yang
et al., 1998; Sumi et al., 1999). Furthermore,
serine/threonine kinases, p21-activated kinase (PAK), and
Rho-associated kinase (ROCK), which are downstream effectors of Rac and
Rho, respectively, directly phosphorylate a threonine residue (Thr-508)
within the activation loop in the kinase domain of LIMK1 and
significantly enhance the kinase activity (Edwards et al.,
1999; Maekawa et al., 1999; Ohashi et al., 2000;
Amano et al., 2001). These observations suggest that both
Rac-PAK and Rho-ROCK signaling pathways can activate LIM-kinases, an
event that in turn induces phosphorylation and inactivation of cofilin.
Considering the significant role of cofilin in actin filament dynamics
and its predicted important functions in diverse cell activities, it is
conceivable that protein kinases other than LIM-kinases are involved in
cofilin phosphorylation and play roles in signaling pathways distinct
from those of LIM-kinases.

Testicular protein kinase 1 (TESK1) is a protein kinase with a unique
structure composed of an N-terminal protein kinase domain and a
C-terminal proline-rich domain (Toshima et al., 1995). TESK1
was named after its higher expression in the testis (Toshima et
al., 1995, 1998), but we recently found its expression in various
tissues and cell lines, albeit at a relatively low level; hence we
assumed that TESK1 has general cellular functions rather than a
specific function in the testis (Toshima et al., 1999). The
protein kinase domain of TESK1 is closely related to those of
LIM-kinases, with ~50% amino acid identity, although their overall
domain structures do differ (Toshima et al., 1995).
Phylogenetic analysis of the protein kinase domains further revealed
that TESK1 and LIM-kinases constitute a novel subfamily within a
serine/threonine kinase family (Toshima et al., 1995). These
observations led to the notion that TESK1 can phosphorylate cofilin/ADF
family proteins and that it plays a role in the actin reorganization,
as do LIM-kinases.

We now provide evidence that TESK1 phosphorylates cofilin and
stimulates the formation of actin stress fibers and focal adhesions. In
contrast to LIM-kinases, the kinase activity of TESK1 is not stimulated
by either ROCK or PAK but can be stimulated by plating cells on a
fibronectin-coated surface. We propose that TESK1 has a role in
integrin-mediated actin cytoskeletal reorganization through the
phosphorylation of cofilin.

In Vivo Kinase Assay

In vivo kinase assay was performed using HA-tagged cofilin or
ADF, as described (Yang et al., 1998). COS-7 cells were
cotransfected with plasmid coding for HA-tagged cofilin, ADF, or
S3A-cofilin, and plasmid for TESK1. Cells were labeled in DMEM
containing 500 μCi/ml [32P]phosphoric acid
for 4 h, washed three times with ice-cold PBS, and suspended in
RIPA buffer. After incubation on ice for 1 h, lysates were
precleared with Protein A-Sepharose (20 μl of 50% slurry) for 3
h at 4°C. The precleared supernatants were incubated with 12CA5
anti-HA-tag antibody (Roche Diagnostics) and Protein A-Sepharose
(20 μl of 50% slurry) overnight at 4°C. After centrifugation, the
immunoprecipitates were washed three times with wash buffer and
subjected to SDS-PAGE in 15 and 9% gels. Proteins were transferred
onto polyvinylidene difluoride membranes. The membrane from a 15% gel
was analyzed by autoradiography to measure
32P-labeled cofilin or ADF, using the BAS1800
Bio-Image Analyzer (Fuji Film) and immunoblotting with
12CA5 anti-HA or anti-Sky-peptide antibody. The membrane from 9% gel
was analyzed by immunoblotting with anti-TESK1
antibody.

Adhesion and Spreading Assay

For cell adhesion assay, 35- or 100-mm culture dishes were
coated overnight at 37°C with 20 μg/ml fibronectin (purchased from
Sigma) or 50 μg/ml poly-l-lysine (Sigma) in TBS buffer
(25 mM Tris-HCl, pH 8.0, 150 mM NaCl) and blocked with 1% bovine serum
albumin (Fraction V, Sigma) in TBS buffer before cells are plated. HeLa
cells (3 × 106 cells) cultured for 24
h in serum-free DMEM were trypsinized, suspended in 5 ml DMEM, then
plated on fibronectin- or poly-l-lysine–coated and bovine
serum albumin-blocked 100-mm dishes. After incubation for 0–60 min at
37°C, adherent cells were washed twice with cold PBS and lysed in
RIPA buffer. Endogenous TESK1 and LIMK1 were immunoprecipitated from
cell lysates, using anti-TESK1 (TK-C21) or anti-LIMK1 antibody (C-10),
and immunoprecipitates were subjected to in vitro kinase reaction. To
determine the level of P-cofilin, cells were lysed with hot SDS buffer
(50 mM Tris-HCl, pH 6.5, 10% glycerol, 2% SDS, 2% 2-mercaptoethanol)
at 95°C for 5 min and sonicated. After centrifugation, supernatants
were subjected to SDS-PAGE and analyzed by
immunoblotting with anti-P-cofilin antibody. For cell
staining, HeLa cells were transfected with plasmids coding for TESK1 or
TESK1(D170A) and cultured for 24 h in serum-free DMEM.
Approximately 2 × 105 cells were
trypsinized, suspended in 1 ml DMEM, and then replated on
fibronectin-coated 35-mm dishes. After incubation for 90 min at 37°C,
cells were washed twice with PBS, fixed in 4% formaldehyde in
phosphate buffer, and costained with TK-C21 anti-TESK1 antibody and
rhodamine-phalloidin or anti-vinculin antibody.

RESULTS

Expression of TESK1 Protein in Various Cell Lines

We previously showed the expression of TESK1 mRNA in various
tissues and cell lines, although the level of expression is lower than
that in the testis (Toshima et al., 1999). To examine the
expression of TESK1 protein in cell lines, lysates from various cell
lines were subjected to immunoprecipitation and immunoblot
analysis, using TK-C21 anti-TESK1 antibody. In our previous studies, we
could not detect the expression of endogenous TESK1 protein in COS-7
cell lysates, under conditions in which we used lysates from 5 ×
105 COS-7 cells (Toshima et al., 1995,
1999). In this study, we therefore used 10 times more cell lysates
(5 × 106 cells), and the
immunoblot membrane was exposed eightfold longer (2 min) to
the ECL detection reagent, compared with the conditions used in the
previous studies. As shown in Figure ​Figure1A,
1A,
the one major immunoreactive band migrating at ~68 kDa, similar to
the calculated mass for TESK1 protein, was detected in various cell
lines, including COS-7 cells, HeLa epithelial carcinoma cells, Rat1A
and Swiss 3T3 fibroblasts, Jurkat T cell leukemia, N1E-115
neuroblastoma, and PC12 pheochromocytoma cells. This band was not
detected when COS-7 cell lysates were immunoprecipitated with preimmune
serum or with anti-TESK1 antibody preincubated with antigenic peptide
(Figure ​(Figure1B),1B), which suggests that the 68-kDa band represents the
endogenously expressed TESK1 protein. The wide expression of TESK1 mRNA
and protein in various tissues and cell lines suggests general cellular
functions of TESK1.

(A) Immunoblot
analysis of endogenous TESK1 protein expressed in various cell lines.
Lysates prepared from approximately 5 × 106 cells of
each cell line were immunoprecipitated with anti-TESK1 antibody
(TK-C21), run on SDS-PAGE, and immunoblotted...

TESK1 Induces the Formation of Stress Fibers and Focal Adhesions

LIM-kinases induce actin reorganization in cultured cells. Because
the kinase domain of TESK1 is similar to those of LIM-kinases, we asked
whether TESK1 can induce changes in actin organization. When HeLa cells
were transfected with plasmids coding for TESK1 and then stained with
rhodamine-phalloidin to visualize actin filaments, marked induction
of actin stress fibers was observed in TESK1-transfected cells,
compared with findings with TESK1-nontransfected cells (Figure
​(Figure2A,2A, top panels). In contrast, expression
of a kinase-inactive mutant of TESK1, TESK1(D170A), in which the
presumptive catalytic residue Asp-170 is replaced by alanine, failed to
induce stress fibers and seemed to partially reduce naturally occurring
stress fibers (Figure ​(Figure2A,2A, middle panels). These results suggest that
TESK1 can induce the formation of actin stress fibers, the function of
which depends on its kinase catalytic activity. As reported (Yang
et al., 1998), expression of LIMK1 induced the actin
reorganization in HeLa cells, but the morphology of polymerized actin
structures induced by LIMK1 was distinct from that induced by TESK1; in
most of the LIMK1-expressing cells, actin filaments accumulated in the
cell periphery (Figure ​(Figure2A,2A, bottom panels). Thus, TESK1 seems to play a
role distinct from that of LIMK1 in actin reorganization. In addition,
we observed that both TESK1 and its kinase-inactive mutant expressed in
HeLa cells were localized diffusely in the cytoplasm, with dense
staining at the perinuclear region; the pattern was distinct from that
of LIMK1, which was enriched in the region of polymerized actin at the
cell periphery (Figure ​(Figure2A).
2A).

The formation of stress fibers is usually accompanied by assembly of
focal adhesions at cell margins. To determine whether TESK1 would
induce focal adhesions, we transfected the TESK1 plasmid into HeLa
cells, and the formation of focal adhesions was visualized by
immunostaining vinculin, a major component of focal adhesions.
Expression of TESK1 significantly induced the formation of focal
adhesions, because vinculin staining was specifically enhanced at the
margins of TESK1-expressing cells (Figure ​(Figure2B,2B, top panels). In contrast,
expression of a kinase-inactive TESK1(D170A) failed to induce focal
adhesions (Figure ​(Figure2B,2B, bottom panels). These findings suggest that TESK1
plays a role in the formation of actin stress fibers and focal
adhesions, both of which depend on its protein kinase activity.

TESK1 Phosphorylates Cofilin and ADF In Vitro and In Vivo

To elucidate the mechanism by which TESK1 induces stress fibers
and focal adhesions, we searched for the kinase substrate(s) for TESK1.
Structural similarity of the kinase domains of TESK1 and LIM-kinases
suggests that the cellular substrate(s) of these kinases may be similar
or even identical. Because LIM-kinases phosphorylate cofilin
specifically at Ser-3, the site of its inactivation, we examined
whether TESK1 could phosphorylate cofilin in vitro and in vivo.
Wild-type TESK1 and its kinase-inactive D170A mutant were expressed in
COS-7 cells, immunoprecipitated, and subjected to in vitro kinase
reaction, using recombinant (His)6-tagged cofilin
as a substrate. As shown in Figure ​Figure3A,
3A,
wild-type TESK1 phosphorylated wild-type cofilin, but not S3A-cofilin,
in which Ser-3 is replaced by alanine. TESK1(D170A) did not
phosphorylate either one. These results suggest that TESK1
phosphorylates cofilin specifically at Ser-3 in vitro. We also examined
the kinase activity of TESK1 toward ADF, a protein closely related to
cofilin. TESK1 phosphorylated ADF to an extent similar to that seen
with cofilin, but not its S3A mutant (Figure ​(Figure3A).3A). Accordingly, TESK1
phosphorylates both cofilin and ADF specifically at Ser-3.

In vitro and in vivo phosphorylation of
cofilin and ADF by TESK1. (A) In vitro kinase assay.
(His)6-tagged wild-type (WT) cofilin, ADF, or their S3A
mutants were incubated with [γ-32P]ATP and Myc-tagged
wild-type (WT) TESK1...

We next asked whether TESK1 could phosphorylate cofilin and ADF in
cultured cells. HA-tagged cofilin or its S3A mutant was expressed with
wild-type or the kinase-inactive form of TESK1 in COS-7 cells, and then
the cells were labeled with
[32P]orthophosphate.
32P-incorporation into cofilin was evident when
coexpressed with wild-type TESK1 but not with TESK1(D170A) (Figure ​(Figure3B,
3B,
left panel). 32P-incorporation into S3A-cofilin
was nil by coexpression with either TESK1 or TESK1(D170A). In a similar
manner, we observed 32P-incorporation into
wild-type ADF but not into its S3A mutant, when they were coexpressed
with TESK1 (Figure ​(Figure3B,3B, right panel). Thus TESK1 can phosphorylate
cofilin and ADF specifically at Ser-3 in vivo as well as in vitro.

Cofilin and S3A-cofilin, when expressed in HeLa cells, induced marked
decrease in rhodamine-phalloidin staining, which was caused by the
actin-binding and -depolymerizing activity of cofilin (Figure ​(Figure3C,3C, left
panels). When TESK1 was coexpressed with cofilin, the decrease in
phalloidin staining induced by cofilin was reversed, and phalloidin
staining of actin filaments was observed (Figure ​(Figure3C,3C, top right panel).
In contrast, the decrease in phalloidin staining induced by
nonphosphorylatable S3A-cofilin was not affected by coexpression with
TESK1 (Figure ​(Figure3C,3C, bottom-right panels). Coexpression with a
kinase-inactive TESK1(D170A) did not reverse the cofilin-induced loss
of phalloidin staining (our unpublished results). These results further
support the idea that TESK1, like LIM-kinases, can phosphorylate
cofilin at Ser-3 and thereby inhibit the actin binding/depolymerizing
activity of cofilin in cultured cells.

Effects of ROCK and PAK on the Kinase Activity of TESK1

Rho and Rac regulate actin reorganization: Rho induces actin
stress fibers and focal adhesions, whereas Rac induces lamellipodia
(Narumiya et al., 1997; Hall, 1998). ROCK and PAK,
downstream effectors of Rho and Rac, respectively, directly
phosphorylate and activate LIM-kinases. Because TESK1 induces stress
fibers and focal adhesions, this kinase may act as a downstream
effector of Rho and ROCK. We therefore tested the effect of Rho-ROCK
pathway activation on the kinase activity of TESK1. When TESK1 was
coexpressed in COS-7 cells with either an active form of Rho (RhoV14)
or an active form of ROCK (ROCKΔ3), no increase in the kinase
activity of TESK1 was observed (our unpublished results). This finding
is in contrast to cases of LIM-kinases, the kinase activities of which
were increased by coexpression with ROCKΔ3 or RhoV14 (Maekawa
et al., 1999; Sumi et al., 1999; Ohashi et
al., 2000; Amano et al., 2001). The kinase activity of
TESK1 was not affected when it was coexpressed with an active form of
Rac (RacV12) or Cdc42 (Cdc42V12) (our unpublished results). In
addition, expression of a kinase-inactive mutant TESK1(D170A) had no
apparent effect on RhoV14- or ROCKΔ3-induced stress fiber formation
or RacV12-induced lamellipodium formation (our unpublished results). In
vitro kinase reaction further revealed that the kinase activity of
TESK1 was not affected by treatment with either ROCKΔ3 or an active
form of PAK (PAKΔN), whereas the kinase activity of LIMK2 was
significantly enhanced by ROCKΔ3 or PAKΔN (Figure
​(Figure4).4). It is noted that TESK1 and
TESK1(D170A) were evidently phosphorylated by PAKΔN (Figure ​(Figure4B,4B, third
panel), but the physiological meaning of this phosphorylation remains
to be determined. Taken together, these results suggest that in
contrast to LIM-kinases, TESK1 is not a downstream effector of either
ROCK or PAK.

ROCK and PAK activate LIMK but not TESK1.
Lysates from COS-7 cells transfected with vector alone (mock) or
plasmids for Myc-tagged TESK1, TESK1(D170A), or LIMK2 were
immunoprecipitated with anti-Myc antibody and incubated with
[γ-...

To further investigate the relationship between the Rho-ROCK
signaling and TESK1-induced actin stress fibers, we examined the
effects of Rho-ROCK inhibitors on TESK1-induced stress fibers. As shown
in Figure ​Figure5A,5A, TESK1-induced stress fibers
were repressed by coexpression with C3 exoenzyme, a
botulinum toxin that specifically inactivates Rho by
ADP-ribosylation (Sekine et al., 1989), or ROCK(KD-IA), a
dominant-negative mutant of ROCK (Ishizaki et al., 1997).
Focal adhesions induced by TESK1 were also repressed by coexpression
with C3 or ROCK(KD-IA) (our unpublished results). Thus, TESK1, albeit
not a direct target of ROCK, does require activity of the Rho-ROCK
signaling pathway for the formation of stress fibers and focal
adhesions.

(A) Suppression of TESK1-induced stress fibers by
C3 or ROCK(KD-IA). HeLa cells were transfected with plasmids for TESK1
with vector alone or plasmids for C3 or ROCK(KD-IA) and stained with
anti-TESK1 antibody (left panels) and rhodamine-phalloidin...

In addition, we also found that coexpression of ROCK(KD-IA) with
TESK1(D170A) in HeLa cells induced an almost complete loss of actin
filaments and remarkable changes in cell morphology (cell shrinkage
leaving process-like structures) (Figure ​(Figure5B).5B). Although ROCK(KD-IA)
alone also induced such shrinking phenotype in 20% of the transfected
cells, coexpression with TESK1(D170A) significantly augmented the ratio
of shrinking cells to 58% (Figure ​(Figure5C).5C). On the other hand, coexpression
of a kinase-inactive mutant of LIMK1, LIMK1(D460A), had no apparent
effect, which further suggests that LIMK1, but not TESK1, acts as a
downstream effector of ROCK and that LIMK1 and TESK1 play distinct
roles in the organization of actin filaments and cell morphology.
Coexpression of wild-type TESK1 with ROCK(KD-IA) reduced the ratio of
shrinking cells. These results suggest that both TESK1 and ROCK have an
important role in maintaining normal cell morphology and adhesion by
supporting the formation of actin stress fibers and focal adhesions.

We next investigated the effects of Y-27632, a specific inhibitor of
ROCK (Uehata et al., 1997), on TESK1-induced actin
reorganization, to determine the short-term effects of ROCK inhibition.
As reported (Uehata et al., 1997), stress fibers induced by
RhoV14 were suppressed by a 30 min treatment of cells with Y-27632.
Similarly, stress fibers in TESK1-transfected cells were reduced by
treatment with Y-27632, but interestingly most of the TESK1-expressing
cells showed polymerized actin structures and vinculin assemblies at
cell peripheries (Figure ​(Figure6,6, A and B). In
TESK1(D170A)-expressing cells, no such structure but rather a
significant loss of actin filaments was observed (Figure ​(Figure6A).6A). TESK1 was
enriched in the region of polymerized actin structures at cell
peripheries in the presence of Y-27632 (Figure ​(Figure6A).6A). Costaining with
anti-TESK1 and anti-vinculin antibodies further revealed colocalization
of TESK1 with vinculin at peripheries of Y-27632–treated cells (Figure
​(Figure6B).6B). Thus, under conditions that ROCK signaling was temporarily blocked
by Y-27632, TESK1 induced polymerized actin structures and vinculin
assemblies that are distinct from stress fibers and focal adhesions, at
the cell periphery. Together with findings that ROCK does not activate
TESK1, these observations suggest that TESK1 has a potential to induce
actin reorganization independently on ROCK.

Effect of Y-27632 on TESK1-induced actin
organization. HeLa cells transfected with plasmids encoding TESK1 or
TESK1(D170A) were treated with 10 μM Y-27632 for 30 min, then fixed
and stained with anti-TESK1 antibody and rhodamine-phalloidin...

Activation of Endogenous TESK1 by Integrin Signaling

During cell adhesion and spreading on the fibronectin-coated
surface, actin stress fibers and focal adhesions are induced by
signaling mediated by integrin receptors (Clark and Brugge,
1995). Because TESK1 can induce assembly of actin stress fibers and
focal adhesions, it may play a role in integrin signaling. We
therefore examined changes in kinase activity of endogenous TESK1
during adhesion and spreading of HeLa cells on fibronectin. Suspended
cells were plated onto fibronectin-coated dishes and cultured under the
serum-free conditions. At indicated times, endogenous TESK1 was
prepared by immunoprecipitation, and its kinase activity was measured
in vitro, using recombinant cofilin as a substrate. The kinase activity
of TESK1 gradually increased with time, reaching a maximum level at 30
min after plating (Figure ​(Figure7A).7A). A longer
plating on fibronectin did not further promote the activity of TESK1.
When we examined the kinase activity of endogenous LIMK1 in HeLa cells
under similar conditions, only a small increase in LIMK1 activity was
observed during cell spreading (Figure ​(Figure7A).7A). The activity of TESK1 was
not stimulated when the cells were plated on a surface coated with
poly-l-lysine (Figure ​(Figure7B).7B). These results suggest that the
kinase activity of TESK1 is up-regulated by fibronectin-induced
stimulation of integrin signaling pathways.

Adhesion to fibronectin increases the kinase
activity of TESK1. (A) HeLa cells were suspended and replated on
fibronectin-coated dishes. At indicated times, cells were lysed, and
endogenous TESK1 and LIMK1 were immunoprecipitated and subjected to in
...

Increase in the Level of Cofilin Phosphorylation by
Integrin Signaling

To determine the level of cofilin phosphorylation after plating
cells on fibronectin, we prepared the antibody that specifically
recognized the phosphorylated form of cofilin (P-cofilin). The
specificity of the antibody was determined by immunoblot
analysis of a two-dimensional gel of COS-7 cell lysates. The antibody
reacted with P-cofilin (and also the phosphorylated form of ADF as a
minor spot) but not with the dephosphorylated form of cofilin (Figure
​(Figure8A).8A). On one-dimensional gels of lysates
of COS-7 cells transfected with plasmids for C-terminally
(His)6-tagged cofilin and ADF, immunoreactive
bands with the sizes corresponding to
(His)6-tagged cofilin and ADF were detected with
anti-P-cofilin antibody, but they disappeared during CIP treatment and
recovered during rephosphorylation reaction with TESK1 (Figure ​(Figure8B).
8B).
These results suggest that the anti-P-cofilin antibody specifically
recognizes the phosphorylated form of cofilin and ADF.

Adhesion to fibronectin increases the level of
phosphorylated form of cofilin. (A) Specificity of anti-P-cofilin
antibody. Two-dimensional gel electrophoresis of lysates of COS-7 cells
was immunoblotted with anti-P-cofilin and anti-cofilin
antibody....

Using this antibody, we examined changes in the level of endogenous
P-cofilin during adhesion and spreading of HeLa cells on
fibronectin-coated dishes. The level of P-cofilin gradually increased
and reached a maximum level at 30 min after plating on fibronectin but
did not change after plating on poly-l-lysine, which
indicates that integrin signaling stimulates cofilin
phosphorylation (Figure ​(Figure8C).8C). We next examined the effects of Y-27632 on
the P-cofilin level before and after plating cells on fibronectin. The
level of P-cofilin markedly decreased in Y-27632–treated suspended
cells but increased gradually after plating on fibronectin in a time
course similar to that of Y-27632–untreated cells (Figure ​(Figure8,8, D and E).
These results suggest that ROCK significantly contributes to the basal
level of cofilin phosphorylation in HeLa cells before plating, but the
integrin-mediated increase in cofilin phosphorylation is
primarily regulated by a pathway(s) not related to ROCK. Similar
patterns of time-dependent changes in TESK1 activation and P-cofilin
level in response to plating cells on fibronectin, together with the
finding that TESK1 kinase activity is independent of ROCK, suggest that
TESK1 but not LIMK is responsible for integrin-mediated cofilin
phosphorylation.

To further examine the role of TESK1 in integrin-mediated
cofilin phosphorylation, we prepared HeLa cells stably expressing
exogenous TESK1 (HeLa/TESK1) and TESK1(D170A)
[HeLa/TESK1(DA)]. The levels of expression of TESK1 and
TESK1(D170A) in these cells were approximately three- to fourfold
higher than the level of endogenous TESK1, as estimated by
immunoblotting (Figure
​(Figure9A).9A). The level of P-cofilin increased
approximately threefold in HeLa/TESK1 cells and decreased to
approximately half in HeLa/TESK1(DA) cells, compared with that in
parental HeLa cells, whereas the levels of total cofilin in these cells
were similar (Figure ​(Figure9A).9A). When HeLa/TESK1 cells were plated onto
fibronectin-coated dishes, the level of P-cofilin increased and reached
a maximum level at 30 min after plating (Figure ​(Figure9B).9B). On the other hand,
the level of P-cofilin in HeLa/TESK1(DA) cells remained low even after
plating on fibronectin (Figure ​(Figure9B).9B). These results further suggest that
TESK1 is involved in cofilin phosphorylation stimulated by
integrin signaling. We also examined the level of P-cofilin in
HeLa cells expressing wild-type LIMK1 (HeLa/LIMK1). The level of
P-cofilin in HeLa/LIMK1 cells was approximately threefold higher than
the level in parental HeLa cells before plating (Figure ​(Figure9D).9D). However,
in contrast to HeLa/TESK1 cells, the level of P-cofilin in HeLa/LIMK1
cells was not changed after cells were plated on fibronectin (Figure ​(Figure9,
9,
D and E). Thus, it is likely that LIMK1 is involved in cofilin
phosphorylation but does not significantly contribute to the increase
in P-cofilin level induced by plating cells on fibronectin. In
addition, treatment with Y-27632 reduced the level of P-cofilin in
HeLa/LIMK1 cells before and after cells were plated on fibronectin but
had no effect on the level of P-cofilin in HeLa/TESK1 cells before and
after plating on fibronectin (Figure ​(Figure9,9, C–E). Taken together these
results suggest that TESK1 but not LIMK1 is involved in
integrin-mediated cofilin phosphorylation during cell spreading
and that integrin-mediated TESK1 activation and cofilin
phosphorylation are mostly independent of ROCK.

In vivo phosphorylation of cofilin by TESK1 is
independent of Rho-ROCK signaling pathway. (A) HeLa cells stably
expressing TESK1 (HeLa/TESK1) or TESK1(D170A) [HeLa/TESK1(DA)] were
lysed, and the lysates were run on SDS-PAGE and analyzed...

To further investigate the role of TESK1 in
integrin-mediated signaling pathways, we next examined the
effect of expression of wild-type or a kinase-inactive form of TESK1 on
integrin-mediated stress fiber and focal adhesion formation.
HeLa cells transfected with plasmids for TESK1 or TESK1(D170A) were
suspended and then replated on fibronectin-coated dishes. When cells
were stained with rhodamine-phalloidin 1.5 h after plating,
stress fibers were markedly enhanced in TESK1-expressing cells (Figure
​(Figure10A,10A, top panel). In contrast, stress
fibers were significantly weakened in TESK1(D170A)-expressing cells,
compared with surrounding nontransfected cells (Figure ​(Figure10A,10A, bottom
panel), which strongly suggests that endogenous TESK1 is involved in
the stress fiber formation induced by integrin signaling. We
also examined the effect of expression of wild-type or a kinase-dead
form of TESK1 on focal adhesion formation by vinculin immunostaining.
Formation of focal adhesions was substantially enhanced in wild-type
TESK1-transfected cells but was reduced in TESK1(D170A)-transfected
cells (Figure ​(Figure10B).10B). As summarized in Figure ​Figure10C,10C, by plating cells on
fibronectin, stress fibers were induced in 79% of the cells expressing
control GFP and in 95% of cells expressing wild-type TESK1, but in
only 44% of cells expressing TESK1(D170A). Similarly, focal adhesions
were induced in 82% of cells expressing control GFP and in 98% of
cells expressing wild-type TESK1, but in only 43% of cells expressing
TESK1(D170A). Cells expressing a kinase-inactive form of LIMK1,
LIMK1(D460A), induced stress fibers and focal adhesions by plating on
fibronectin, to an extent seen with cells expressing control GFP (our
unpublished results). Suppression of stress fibers and focal adhesions
by TESK1(D170A) but not by LIMK1(D460A) strongly suggests that
endogenous TESK1 but not LIMK1 plays an important role in the
integrin-mediated signaling pathway to induce stress fibers and
focal adhesions.

Effects of TESK1 and TESK1(D170A) on
fibronectin-induced stress fibers and focal adhesions. HeLa cells were
transfected with plasmid coding for TESK1 or TESK1(D170A) and cultured
for 24 h in serum-free medium. Cells were trypsinized and replated
on...

DISCUSSION

Cofilin Phosphorylation by TESK1 and LIM-kinases

Cofilin plays an essential role in actin filament dynamics
by enhancing depolymerization and severance of actin filaments (Bamburg
et al., 1999). These activities of cofilin are abolished by
phosphorylation at Ser-3; therefore, phosphorylation/dephosphorylation
of cofilin at Ser-3 is regarded as one of the important mechanisms for
regulating cofilin activities and actin filament dynamics. LIM-kinases
were shown to be responsible enzymes for cofilin phosphorylation (Arber
et al., 1998; Yang et al., 1998). Here we provide
evidence that TESK1 also has an ability to phosphorylate cofilin
specifically at Ser-3 in vitro and in vivo and induce actin
reorganization by phosphorylating cofilin. Thus, these findings suggest
that cofilin phosphorylation in living cells is regulated by at least
two pathways in which distinct types of protein kinases, LIM-kinases
and TESK1, are involved. Considering the essential role of cofilin in
actin filament dynamics, these kinases probably play important roles in
regulating actin cytoskeletal remodeling and thereby in many cell
activities, including cell motility, adhesion, and cytokinesis, by
phosphorylating and inactivating cofilin. Most interestingly, we found
that TESK1 is stimulated by an integrin-mediated signaling
pathway but not by either ROCK or PAK, in contrast to LIM-kinases that
are stimulated by Rho-ROCK and Rac-Pak pathways (Figure
​(Figure11).11). Together with the total difference
in extracatalytic structures, these results strongly suggest that
LIM-kinases and TESK1, although they commonly phosphorylate cofilin,
are regulated in different ways and play distinct roles in actin
reorganization in living cells.

Proposed scheme for multiple pathways for cofilin
phosphorylation. TESK1 is activated downstream of integrin
signaling pathways, whereas LIM-kinases are activated downstream of
Rho-ROCK and Rac-PAK pathways. See text for details.

TESK1-induced Stress Fibers and Rho-ROCK Signaling Pathway

Rho and its downstream ROCK play a key role in the formation of
stress fibers and focal adhesions (Ridley and Hall, 1992; Leung
et al., 1996; Amano et al., 1997; Ishizaki
et al., 1997). ROCK induces stress fibers by increasing
myosin light chain (MLC) phosphorylation through phosphorylating and
inactivating MLC phosphatase and thereby increasing actomyosin-based
contractility (Kimura et al., 1996). ROCK also
phosphorylates and activates LIM-kinases, events that lead to
phosphorylation and inactivation of cofilin and actin filament
stabilization (Maekawa et al., 1999). Because TESK1
stimulates the formation of stress fibers and focal adhesions, we
extensively examined the relationships between TESK1 and the Rho-ROCK
signaling pathway and obtained evidence that distinct from LIM-kinases,
TESK1 is not downstream of ROCK. However, one cannot exclude the
possibility that the kinase activity of TESK1 might be regulated by
downstream effectors of Rho other than ROCK, such as rhophilin,
rhotekin, and p160mDia, because in our assay systems we may overlook
activation of TESK1 in cultured cells if activated by noncovalent
association of activator proteins.

TESK1-induced stress fibers and focal adhesions were repressed by
coexpression of C3 exoenzyme or ROCK(KD-IA), which suggests that the
Rho-ROCK signaling pathway is required for the formation of stress
fibers and focal adhesions, although ROCK does not stimulate TESK1.
Most likely, the ROCK-induced increase in MLC phosphorylation and
actomyosin contractility is required for formation of stress fibers and
focal adhesions (Kimura et al., 1996). Interestingly,
Y-27632 treatment of TESK1-expressing cells led to the formation of
lamellipodium-like actin organization and vinculin-assembled structures
at cell margins, in place of stress fibers and focal adhesions. TESK1
therefore seems to have the potential to induce distinct patterns of
actin organization, under conditions that ROCK signaling is abrogated.
Several studies suggest that Rho and Rac mutually antagonize cellular
activity, and the balance between Rho and Rac activity in cells
determines the patterns of actin organization and substrate contact
site morphology (Hirose et al., 1998; Rottner et
al., 1999; Sander et al., 1999). Thus, it could be that
lamellipodium-like actin organization and vinculin assembly in
TESK1-expressing cells treated with Y-27632 are brought about by the
preference of Rac activity against Rho activity as the result of
inhibition of ROCK by Y-27632 treatment.

We also found that coexpression of TESK1(D170A) and ROCK(KD-IA) induces
an almost complete loss of actin stress fibers and shrinking cell
morphology. Because TESK1(D170A) and ROCK(KD-IA) are thought to
function as dominant-negative forms against endogenous TESK1 and ROCK,
respectively, both TESK1 and ROCK probably play an important role in
maintaining cell morphology and cell adhesion by retaining certain
levels of actin stress fibers and focal adhesions. Manser et
al. (1997) reported a similar shrinking cell morphology induced by
expression of an active form of PAK. Such morphological change may be
due to the PAK activity to phosphorylate and inactivate MLC-kinase
(Sanders et al., 1999), which leads to loss of
actomyosin-based contractility and dissolution of stress fibers and
focal adhesions, an event opposite that induced by ROCK/TESK1
activation. Thus, we assume that formation of stress fibers and focal
adhesions are oppositely regulated by ROCK/TESK1 and PAK, and
inhibition of both ROCK and TESK1 or excessive activation of PAK
results in similar shrinking cell morphology.

Roles of TESK1 in Integrin-mediated Signaling Pathway

Integrins bind to ECM proteins such as fibronectin and
transduce signals to control cell survival, proliferation,
differentiation, and migration (Clark and Brugge, 1995; Giancotti and
Ruoslahti, 1999). On binding to ECM proteins, integrins become
clustered and form large protein complexes known as focal adhesions,
through which integrins link to actin filaments (Burridge
et al., 1997). Previous studies identified a number of
proteins that are assembled into focal adhesions on integrin
stimulation, but little is known about signaling pathways of
integrin-mediated actin reorganization. In the present study we
have found that the kinase activity of TESK1 as well as the level of
cofilin phosphorylation are elevated by plating cells on fibronectin.
Time courses of changes in TESK1 activity and P-cofilin level after
plating are similar, and the level of P-cofilin increased in cells
stably expressing TESK1 but decreased in cells expressing TESK1(D170A).
We have also found that wild-type TESK1 enhanced and a kinase-inactive
form of TESK1 suppressed the fibronectin-induced formation of stress
fibers and focal adhesions. Taken together, these results suggest that
TESK1 plays a key role in cofilin phosphorylation and actin
reorganization stimulated by integrin signaling. In contrast,
only a small increase in the kinase activity of LIMK1 was detected
after plating cells on fibronectin, and a kinase-inactive form of LIMK1
had no apparent effect on integrin-mediated stress fiber and
focal adhesion formation. Furthermore, the level of P-cofilin in
HeLa/LIMK1 cells increased before plating but did not change after
plating cells on fibronectin. Accordingly, it is presumable that LIMK
does not significantly contribute to integrin-mediated cofilin
phosphorylation and actin reorganization.

Treatment with Y-27632 significantly reduced the level of P-cofilin
before plating but had no apparent effect on the fibronectin-stimulated
cofilin phosphorylation, which indicates that ROCK is involved in
maintaining the basal level of P-cofilin but has little effect on
integrin-mediated cofilin phosphorylation. In addition,
treatment with Y-27632 reduced the level of P-cofilin in HeLa/LIMK1
cells before and after plating cells on fibronectin but did not affect
the level of P-cofilin in HeLa/TESK1 cells. Together with the finding
that ROCK activates LIMK1 but not TESK1 in in vitro and in vivo
reactions, these results suggest that TESK1 principally contributes to
the integrin-mediated cofilin phosphorylation, which is
independent of ROCK, whereas LIMK1 contributes to cofilin
phosphorylation before plating that is dependent on ROCK activity.

Previous studies implicated Rho and Rac in integrin-mediated
cell adhesion and spreading (Clark et al., 1998; Price
et al., 1998; Ren et al., 1999). Rho was
significantly activated by plating cells on fibronectin when cells were
maintained in 1% serum, but its activation was very modest under
serum-free conditions (Ren et al., 1999). Only a small
increase in LIMK1 activity and Y-27632 insensitivity of cofilin
phosphorylation after cells were plated on fibronectin in our assays
may be explained by our assay conditions in which serum was depleted.
It could be that under serum-supplemented conditions TESK1 and Rho
signals cooperatively function in integrin-mediated stress
fiber and focal adhesion formation. In fibroblasts, plating cells on
fibronectin led to the rapid activation of PAK with the maximal
activity at 5–10 min after plating (Price et al., 1998). If
this is also the case in HeLa cells, PAK activation may contribute to
the early phase of integrin-mediated cofilin phosphorylation
through activation of LIMK, but activation of LIMK1 was barely
detectable at 15 min after plating in our assay. Further studies are
needed to elucidate the role of the Rac-PAK pathway in
integrin-mediated cofilin phosphorylation in respective cells.

The C-terminal region of TESK1 is rich in proline residues and contains
several ProX-X-Pro motifs, known to be recognized by SH3 domains. TESK1
may be localized and activated at the sites of cell adhesion by
interaction with SH3-containing focal adhesion proteins, such as Crk,
Nck, and CAS, through its C-terminal proline-rich region. Focal
adhesion proteins, such as CAS and paxillin, are phosphorylated on
serine, threonine, and tyrosine residues integrin during
stimulation (Schlaepfer et al., 1997; Brown et
al., 1998). Because TESK1 is activated by integrin
stimulation, TESK1 may play a role in actin reorganization and focal
adhesion formation by phosphorylating these focal adhesion proteins, in
addition to phosphorylating cofilin.

Physiological Roles of TESK1

TESK1 mRNA is predominantly expressed in testicular germ cells at
stages of late pachytene spermatocytes to round spermatids, which
suggests a role of TESK1 in spermatogenesis (Toshima et al.,
1995, 1998). However, the actual function of TESK1 in testicular germ
cells remains unknown. Drosophila null mutants of the
center divider (cdi) gene, which encodes an
orthologue of TESK1, are larval lethal, suggesting an important role
for the cdi gene product in fly development (Matthews and
Crews, 1999). The cdi gene is prominently expressed in
midline cells of the fly embryonic CNS. Because we observed that TESK1
gene is expressed in specific regions of the mouse embryonic CNS (our
unpublished data), there may be functional relationships between fly
cdi and mammalian TESK1 during neuronal development. On the
other hand, whether the cdi gene is expressed in fly testes
or whether mutation of the cdi gene affects fly
spermatogenesis remains to be determined. Using Drosophila
genetics to explore the functions of TESK1 in spermatogenesis and
neurogenesis is a challenging theme.

Drosophila twinstar (tsr) mutants, in which
expression of the tsr gene encoding a Drosophila
cofilin orthologue is reduced, are lethal in late larval or pupal
stages (Gunsalus et al., 1995). Cytological studies revealed
frequent failures in cytokinesis in larval neuroblasts and testicular
meiotic cells. Mutant spermatocytes exhibited delayed centrosome
migration and a defect in contractile ring disassembly of two meiotic
cell divisions (Gunsalus et al., 1995). A similar phenotype
was seen in testes treated with cytochalasin B, an inhibitor of actin
polymerization. These phenotypes in mutant spermatocytes are regarded
as the aberrant actin cytoskeletal reorganization, and the properly
regulated actin assembly and disassembly are likely important for
normal centrosome migration and normal contractile ring formation and
dissolution during cytokinesis. TESK1 in testes may play a role in
these processes during spermatogensis by regulating the activity of
cofilin. On the basis of activity of TESK1 to phosphorylate cofilin, it
seems appropriate to determine physiological functions of TESK1, as
related to actin cytoskeletal reorganization.

In conclusion, our evidence shows that cofilin phosphorylation is
regulated by at least two distinct types of protein kinases:
LIM-kinases and TESK1. Although LIM-kinases are activated by Rac-PAK
and Rho-ROCK signaling pathways, TESK1 is stimulated by
integrin-mediated signaling pathways and significantly
contributes integrin-mediated cofilin phosphorylation and actin
reorganization (Figure ​(Figure11).11). Our findings will provide new insights into
the integrin-mediated signaling pathways to induce actin
cytoskeletal remodeling and focal adhesion formation.

ACKNOWLEDGMENTS

We thank Dr. Saburo Aimoto (Osaka University) for phosphopeptide
synthesis, Dr. Takashi Obinata (Chiba University) for providing MAB-22
anticofilin monoclonal antibody, Dr. Kyoko Ohashi (Tohoku
University) for two-dimensional gel analysis, and Dr. Yukio Fujiki
(Kyushu University) for advice and encouragement. This work was
supported by research grants from the Ministry of Education, Science,
Sports, and Culture of Japan, and the Japan Society of the Promotion of
Science Research for the Future, and grants from Naito Foundation,
Welfide Foundation, and Uehara Memorial Foundation (to K.M.).

Leung T, Chen X-Q, Manser E, Lim L. The p160 RhoA-binding kinase ROKα is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol. 1996;16:5313–5327.[PMC free article][PubMed]